The accomplishments of the section are: 1. We discovered that ingestion of a second uninfected blood meal by Leishmania-infected sand flies triggers parasite dedifferentiation into a new stage, we named retroleptomondas , this new parasite stage multiplies greatly in the insect enhancing disease transmission. Sand flies, similar to most vectors, take multiple blood meals during their lifetime. The effect of subsequent blood meals on pathogens developing in the vector and their impact on disease transmission have never been examined. We demonstrated that ingestion of a second uninfected blood meal by Leishmania-infected sand flies triggers dedifferentiation of metacyclic promastigotes to a leptomonad-like stage, the retroleptomonad promastigote by a process we are calling Reverse metacyclogenesis. During this process retroleptomonad promastigotes rapidly multiply and differentiate to metacyclic promastigotes enhancing sand fly infectiousness. These findings place readily available blood sources as a critical element in transmission and propagation of vector-borne pathogens. 2. The sand fly gut microbiota that is egested by Leishmania-infected sand ies prime the host inammasome and is critical for parasite visceralization. We demonstrate that gut microbes from the sand y are egested into host skin alongside Leish-mania parasites. The egested microbes trigger the inammasome, leading to a rapid production of inter-leukin-1b (IL-1b), which sustains neutrophil inltration. Reducing midgut microbiota by pretreatment of Leishmania-infected sand ies with antibiotics or neutralizing the effect of IL-1b in bitten mice abrogates neutrophil recruitment. These early events are associated with impairment of parasite visceralization, indicating that both gut microbiota and IL-1b are important for the establishment of Leishmania infections. Considering that arthropods harbor a rich microbiota, its potential egestion after bites may be a shared mechanism that contributes to severity of vector-borne disease. 3. Immunization with the sand fly salivary proteins LuloHya and Lundep, protects animals against Leishmania major infection. A vaccination experiment demonstrated that LuloHya and Lundep confer protective immunity against cutaneous leishmaniasis using the Lu. longipalpis-Leishmania major combination as a model. Animals (C57BL/6) immunized with LuloHya or Lundep showed minimal skin damage while lesions in control animals remained ulcerated. This protective immunity was abrogated when B-cell-deficient mice were used indicating that antibodies against both proteins play a significant role for disease protection. Rabbit-raised anti-LuloHya antibodies completely abrogated hyaluronidase activity in vitro. Moreover, in vivo experiments demonstrated that blocking LuloHya with specific antibodies interferes with sand fly blood feeding. This work highlights the relevance of vector salivary components in blood feeding and parasite transmission and further suggests the inclusion of these salivary proteins as components for an anti-Leishmania vaccine. 4.- Pre-clinical evaluation of an innovative multivalent vaccine for human visceral leishmaniasis that combines Leishmania and sand fly salivary antigens. We optimized and assessed in vivo the safety and immunogenicity of an innovative vaccine candidate against human visceral leishmaniasis (VL), consisting of Virus-Like Particles (VLP) loaded with three different recombinant proteins (LJL143 from Lutzomyia longipalpis saliva as the vector-derived (VD) component, and KMP11 and LeishF3+, as parasite-derived (PD) antigens) and adjuvanted with GLA-SE, a TLR4 agonist. No apparent adverse reactions were observed during the experimental time-frame, which together with the normal hematological parameters detected seems to point to the safety of the formulation. Furthermore, measurements of antigen-specific cellular and humoral responses, generally higher in immunized versus control groups, confirmed the immunogenicity of the vaccine formulation. Interestingly, the immune responses against the VD protein were reproducibly more robust than those elicited against leishmanial antigens, and were apparently not caused by immunodominance of the VD antigen. Remarkably, priming with the VD protein alone and boosting with the complete vaccine candidate contributed towards an increase of the immune responses to the PD antigens, assessed in the form of increased ex vivo CD4+ and CD8+ T cell proliferation against both the PD antigens and total Leishmania antigen (TLA). 5.- Testing a Leishmania parasite subunit vaccine using Leishmania donovani infected sand flies. Incorporating natural challenge models into vaccine development strategies may increase the accuracy of predicting efficacy under field conditions. Until recently, however, there was no natural challenge model available for the evaluation of vaccine candidates against visceral leishmaniasis. At the LMVR we developed a robust natural model of visceral leishmaniaisis in hamsters using L. longipalpis sand flies infected with Leishmania donovani. Using this natural model we tested a next generation candidate, LEISH-F3+ in collaboration with the Infectious Disease Research Institute. Prophylactic immunization with LEISH-F3+ formulated with glucopyranosyl lipid A adjuvant in stable emulsion significantly reduced both Leishmania infantum and L. donovani burdens in needle challenge mouse models of infection. Importantly, the data obtained in these infection models were validated by the ability of LEISH-F3+/glucopyranosyl lipid A adjuvant in stable emulsion to induce significant protection in hamsters, a model of both infection and disease, following challenge by L. donovani-infected Lutzomyia longipalpis sand flies, a natural vector. This is an important demonstration of vaccine protection against visceral leishmaniasis using a natural challenge model.